Improving the study of RNA dynamics through advances in RNA-seq with metabolic labeling and nucleotide-recoding chemistry.

RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and unlabeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U-containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR-seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.

Group Concept Mapping Conceptualizes High-Quality Care for Long-Stay Pediatric Intensive Care Unit Patients and Families.

OBJECTIVE: To describe and conceptualize high-quality care for long-stay pediatric intensive care unit (PICU) patients using group concept mapping (GCM). STUDY DESIGN: We convened an expert panel to elucidate domains of high-quality care for this growing patient population for which transitory care models fail to meet their needs. Thirty-one healthcare professionals and 7 parents of patients with previous prolonged PICU hospitalizations comprised a diverse, interprofessional multidisciplinary panel. Participants completed the prompt "For PICU patients and families experiencing prolonged lengths of stay, high quality care from the medical team includes ______", with unlimited free text responses. Responses were synthesized into individual statements, then panelists sorted them by idea similarity and rated them by perceived importance. Statement analysis using GCM software through GroupWisdom generated nonoverlapping clusters representing domains of high-quality care. RESULTS: Participants submitted 265 prompt responses representing 313 unique ideas, resulting in 78 final statements for sorting and rating. The resultant cluster map best representing the data contained 8 domains: (1) Family-Centered Care and Shared Decision Making, (2) Humanizing the Patient, (3) Clinician Supports and Resources, (4) Multidisciplinary Coordination of Care, (5) Family Well-Being, (6) Anticipatory Guidance and Care Planning, (7) Communication, and (8) Continuity of Care. CONCLUSIONS: GCM empowered a panel of healthcare professionals and parents to explicitly describe and conceptualize high-quality care for patients and families experiencing prolonged PICU stays. This information will aid the effort to address shortcomings of transitory PICU care models.

Continuity Strategies for Long-Stay PICU Patients: Consensus Statements From the Lucile Packard Foundation PICU Continuity Panel.

OBJECTIVES: To develop consensus statements on continuity strategies using primary intensivists, primary nurses, and recurring multidisciplinary team meetings for long-stay patients (LSPs) in PICUs. PARTICIPANTS: The multidisciplinary Lucile Packard Foundation PICU Continuity Panel comprising parents of children who had prolonged PICU stays and experts in several specialties/professions that care for children with medical complexity in and out of PICUs. DESIGN/METHODS: We used modified RAND Delphi methodology, with a comprehensive literature review, Delphi surveys, and a conference, to reach consensus. The literature review resulted in a synthesized bibliography, which was provided to panelists. We used an iterative process to generate draft statements following panelists' completion of four online surveys with open-ended questions on implementing and sustaining continuity strategies. Panelists were anonymous when they voted on revised draft statements. Agreement of 80% constituted consensus. At a 3-day virtual conference, we discussed, revised, and re-voted on statements not reaching or barely reaching consensus. We used Grading of Recommendations Assessment, Development, and Evaluation to assess the quality of the evidence and rate the statements' strength. The Panel also generated outcome, process, and balancing metrics to evaluate continuity strategies. RESULTS: The Panel endorsed 17 consensus statements in five focus areas of continuity strategies (Eligibility Criteria, Initiation, Standard Responsibilities, Resources Needed to Implement, Resources Needed to Sustain). The quality of evidence of the statements was low to very low, highlighting the limited evidence and the importance of panelists' experiences/expertise. The strength of the statements was conditional. An extensive list of potential evaluation metrics was generated. CONCLUSIONS: These expert/parent-developed consensus statements provide PICUs with novel summaries on how to operationalize, implement, and sustain continuity strategies for LSP, a rapidly growing, vulnerable, resource-intensive population in PICUs.

Immune markers are associated with cognitive performance in a multiethnic cohort: The Northern Manhattan Study.

OBJECTIVE: To determine whether immune protein panels add significant information to correlates of cognition. BACKGROUND: Immune mechanisms in vascular cognitive aging are incompletely characterized. DESIGN/METHODS: A subsample of the prospective Northern Manhattan Study underwent detailed neuropsychological testing. Cognitive scores were converted into Z-scores and categorized into four domains (memory, language, processing speed, and executive function) based on factor analysis. Blood samples were analyzed using a 60-plex immunoassay. We used least absolute shrinkage and selection operator (LASSO) procedures to select markers and their interactions independently associated with cognitive scores. Linear regression models assessed cross-sectional associations of known correlates of cognition with cognitive scores, and assessed model fit before and after addition of LASSO-selected immune markers. RESULTS: Among 1179 participants (mean age 70 ± 8.9 years, 60% women, 68% Hispanic), inclusion of LASSO-selected immune markers improved model fit above age, education, and other risk factors (p for likelihood ratio test < 0.005 for all domains). C-C Motif Chemokine Ligand 11 (CCL 11, eotaxin), C-X-C Motif Chemokine Ligand 9 (CXCL9), hepatocyte growth factor (HGF), and serpin E1 (plasminogen activator inhibitor-1) were associated with each of the domains and with overall cognitive function. Immune marker effects were comparable to conventional risk factors: for executive function, each standard deviation (SD) increase in CCL11 was associated with an effect equivalent to aging three years; for memory, HGF had twice the effect of aging. CONCLUSIONS: Immune markers associate with cognitive function in a multi-ethnic cohort. Further work is needed to validate these findings and determine optimal treatment targets.

Race/Ethnic Disparities in Mild Cognitive Impairment and Dementia: The Northern Manhattan Study.

BACKGROUND: Variability in dementia rates across racial and ethnic groups has been estimated at 60%. Studies suggest disparities in Caribbean Hispanic and Black populations, but community-based data are limited. OBJECTIVE: Estimate the prevalence of mild cognitive impairment (MCI) and dementia in the racially and ethnically diverse community-based Northern Manhattan Study cohort and examine sociodemographic, vascular risk factor, and brain imaging correlates. METHODS: Cases of MCI and dementia were adjudicated by a team of neuropsychologists and neurologists and prevalence was estimated across race/ethnic groups. Ordinal proportional odds models were used to estimate race/ethnic differences in the prevalence of MCI or dementia adjusting for sociodemographic variables (model 1), model 1 plus potentially modifiable vascular risk factors (model 2), and model 1 plus structural imaging markers of brain integrity (model 3). RESULTS: There were 989 participants with cognitive outcome determinations (mean age 69±9 years; 68% Hispanic, 16% Black, 14% White; 62% women; mean (±SD) follow-up five (±0.6) years). Hispanic and Black participants had greater likelihood of MCI (20%) and dementia (5%) than White participants accounting for age and education differences. Hispanic participants had greater odds of MCI or dementia than both White and Black participants adjusting for sociodemographic variables, vascular risk factors, and brain imaging factors. White matter hyperintensity burden was significantly associated with greater odds of MCI or dementia (OR = 1.3, 1.1 to 1.6), but there was no significant interaction by race/ethnicity. CONCLUSION: In this diverse community-based cohort, cross-sectional data revealed significant race/ethnic disparities in the prevalence of MCI and dementia. Longer follow-up and incidence data are needed to further clarify these relationships.

Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design.

Riboswitches are mRNA domains that make gene-regulatory decisions upon binding their cognate ligands. Bacterial riboswitches that specifically recognize 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP) and 5'-triphosphate (ZTP) regulate genes involved in folate and purine metabolism. Now, we have developed synthetic ligands targeting ZTP riboswitches by replacing the sugar-phosphate moiety of ZMP with various functional groups, including simple heterocycles. Despite losing hydrogen bonds from ZMP, these analogs bind ZTP riboswitches with similar affinities as the natural ligand, and activate transcription more strongly than ZMP in vitro. The most active ligand stimulates gene expression ∼3 times more than ZMP in a live Escherichia coli reporter. Co-crystal structures of the Fusobacterium ulcerans ZTP riboswitch bound to synthetic ligands suggest stacking of their pyridine moieties on a conserved RNA nucleobase primarily determines their higher activity. Altogether, these findings guide future design of improved riboswitch activators and yield insights into how RNA-targeted ligand discovery may proceed.

Employing a ZTP Riboswitch to Detect Bacterial Folate Biosynthesis Inhibitors in a Small Molecule High-Throughput Screen.

Various riboswitch classes are being discovered that precisely monitor the status of important biological processes, including metabolic pathway function, signaling for physiological adaptations, and responses to toxic agents. Biochemical components for some of these processes might make excellent targets for the development of novel antibacterial molecules, which can be broadly sought by using phenotypic drug discovery (PDD) methods. However, PDD data do not normally provide clues regarding the target for each hit compound. We have developed and validated a robust fluorescent reporter system based on a ZTP riboswitch that identifies numerous folate biosynthesis inhibitors with high sensitivity and precision. The utility of the riboswitch-based PDD strategy was evaluated using Escherichia coli bacteria by conducting a 128 310-compound high-throughput screen, which identified 78 sulfanilamide derivatives among the many initial hits. Similarly, representatives of other riboswitch classes could be employed to rapidly match antibacterial hits with the biological processes they target.

Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure.

Riboswitches are naturally occurring RNA aptamers that regulate gene expression by binding to specific small molecules. Riboswitches control the expression of essential bacterial genes and are important models for RNA-small molecule recognition. Here, we report the discovery of a class of synthetic small molecules that bind to PreQ1 riboswitch aptamers. These molecules bind specifically and reversibly to the aptamers with high affinity and induce a conformational change. Furthermore, the ligands modulate riboswitch activity through transcriptional termination despite no obvious chemical similarity to the cognate ligand. X-ray crystallographic studies reveal that the ligands share a binding site with the cognate ligand but make different contacts. Finally, alteration of the chemical structure of the ligand causes changes in the mode of RNA binding and affects regulatory function. Thus, target- and structure-based approaches can be used to identify and understand the mechanism of synthetic ligands that bind to and regulate complex, folded RNAs.

Measuring RNA-Ligand Interactions with Microscale Thermophoresis.

In recent years, there has been dramatic growth in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA-small molecule interactions. While a host of techniques exist to measure RNA-small molecule interactions, many of these have drawbacks that make them intractable for routine use and are often not broadly applicable. A newer technology called microscale thermophoresis (MST), which measures the directed migration of a molecule and/or molecule-ligand complex along a temperature gradient, can be used to measure binding affinities using very small amounts of sample. The high sensitivity of this technique enables measurement of affinity constants in the nanomolar and micromolar range. Here, we demonstrate how MST can be used to study a range of biologically relevant RNA interactions, including peptide-RNA interactions, RNA-small molecule interactions, and displacement of an RNA-bound peptide by a small molecule.

Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays.

The identification of small molecules that bind to and perturb the function of microRNAs is an attractive approach for the treatment for microRNA-associated pathologies. However, there are only a few small molecules known to interact directly with microRNAs. Here, we report the use of a small molecule microarray (SMM) screening approach to identify low molecular weight compounds that directly bind to a pre-miR-21 hairpin. Compounds identified using this approach exhibit good affinity for the RNA (ranging from 0.8-2.0 µM) and are not composed of a polycationic scaffold. Several of the highest affinity compounds inhibit Dicer-mediated processing, while in-line probing experiments indicate that the compounds bind to the apical loop of the hairpin, proximal to the Dicer site. This work provides evidence that small molecules can be developed to bind directly to and inhibit miR-21.

The Emerging Role of RNA as a Therapeutic Target for Small Molecules.

Recent advances in understanding different RNAs and unique features of their biology have revealed a wealth of information. However, approaches to identify small molecules that target these newly discovered regulatory elements have been lacking. The application of new biochemical screening and design-based technologies, coupled with a resurgence of interest in phenotypic screening, has resulted in several compelling successes in targeting RNA. A number of recent advances suggest that achieving the long-standing goal of developing drug-like, biologically active small molecules that target RNA is possible. This review highlights advances and successes in approaches to targeting RNA with diverse small molecules, and the potential for these technologies to pave the way to new types of RNA-targeted therapeutics.

Mothers' and fathers' differential expectancies and behaviors: parent x child gender effects.

In 3 studies using 6 subscales, the authors investigated (a) others' parenting expectancies for mothers and fathers and (b) parents' reports of the frequency of their parenting behaviors with their 3- to 6-year-old sons and daughters. Mothers rated higher for physical care and emotional support than did fathers, and mothers reported engaging in the parenting behaviors assessed more frequently than did fathers across subscales. In each study, the authors obtained Parent x Child Gender interactions only for personal-interaction parenting (e.g., hugging their child) and found the lowest scores for fathers with daughters. These findings and the relations between parents' ratings of appropriateness and of their own behaviors support the view that gender-based expectancies prescribe gender differences in parenting.

Adult-onset Alexander disease with progressive ataxia and palatal tremor.

A novel glial fibrillary acidic protein (GFAP) mutation, Y257C, is reported in a patient with adult-onset Alexander disease. This is the oldest reported case with confirmation of a GFAP mutation. Onset was late in the sixth decade. Genetic analysis of the GFAP gene is recommended in cases of progressive ataxia and palatal tremor.